Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (2): 344-357.DOI: 10.3969/j.issn.1004-1524.20230318
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Cloning and expression analysis of FLS gene of flavonol synthetase in Lilium auratum and L.speciosum var. gloriosoides
LIU Xiaolin(
), SUN Tingting, YANG Jie, HE Hengbin*()
- Beijing Key Laboratory of Ornamental Plants Germplasm Innovation and Molecular Breeding, National Engineering Research Center for Floriculture, Beijing Laboratory of Urban and Rural Ecological Environment, College of Landscape Architecture, Beijing Forestry University, Beijing 100083,China
-
Received:2023-03-10Online:2024-02-25Published:2024-03-05
CLC Number:
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LIU Xiaolin, SUN Tingting, YANG Jie, HE Hengbin. Cloning and expression analysis of FLS gene of flavonol synthetase in Lilium auratum and L.speciosum var. gloriosoides[J]. Acta Agriculturae Zhejiangensis, 2024, 36(2): 344-357.
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Fig.1 Experimental materials A, L. auratum; B, L. speciosum var. gloriosoides; C, Four-stage materials of L. auratum; D, Four-stage materials of L. speciosum var. gloriosoides.
Fig.1 Experimental materials A, L. auratum; B, L. speciosum var. gloriosoides; C, Four-stage materials of L. auratum; D, Four-stage materials of L. speciosum var. gloriosoides.
Table 1 Primer sequences
| 引物名称 Primer name | 引物序列Primer sequence(5'-3') |
|---|---|
| EF(F) | GGCACTAACTCGCTCCTTCTG |
| EF(R) | GGATTGAGAGGAGCTTCGGTGAGA |
| TIP41(F) | CGAAGCCAGAAACGGAGAAGAAT |
| TIP41(R) | GGGTAGGGTGGATTGGGAAGA |
| FLS(Clone)F | ATGTCTACCCGACATCACCTTG |
| FLS(Clone)R | CTATTATTGTGGAAGTTTGTTAATC |
| FLS(sub-clone)F | CCAAATCGACTCTAGAATGTCTACCCGACATCACCTTG |
| FLS(sub-clone)R | TACCGGATCCACTAGTTTGTGGAAGTTTGTTAATCTTGC |
| FLS(qRT-PCR)F | ACTTAGCACTTGGGGTGGTG |
| FLS(qRT-PCR)R | CGTCCCATTGCTCAAAATCT |
| FLS(RT-PCR)F | GAGGAGAAGGAGGCGATAGC |
| FLS(RT-PCR)R | CGTCTAACCCTAAACCAACTG |
Table 1 Primer sequences
| 引物名称 Primer name | 引物序列Primer sequence(5'-3') |
|---|---|
| EF(F) | GGCACTAACTCGCTCCTTCTG |
| EF(R) | GGATTGAGAGGAGCTTCGGTGAGA |
| TIP41(F) | CGAAGCCAGAAACGGAGAAGAAT |
| TIP41(R) | GGGTAGGGTGGATTGGGAAGA |
| FLS(Clone)F | ATGTCTACCCGACATCACCTTG |
| FLS(Clone)R | CTATTATTGTGGAAGTTTGTTAATC |
| FLS(sub-clone)F | CCAAATCGACTCTAGAATGTCTACCCGACATCACCTTG |
| FLS(sub-clone)R | TACCGGATCCACTAGTTTGTGGAAGTTTGTTAATCTTGC |
| FLS(qRT-PCR)F | ACTTAGCACTTGGGGTGGTG |
| FLS(qRT-PCR)R | CGTCCCATTGCTCAAAATCT |
| FLS(RT-PCR)F | GAGGAGAAGGAGGCGATAGC |
| FLS(RT-PCR)R | CGTCTAACCCTAAACCAACTG |
Table 2 Gene information of system sequence alignment
| 拉丁名Latin name | 登录号Login ID | 基因名Gene name |
|---|---|---|
| Allium cepa L. | AAO63023.1 | AcFLS(H6) |
| A. cepa L. | AQR58516.1 | AcFLS (HRB) |
| Arabidopsis thaliana (L.) Heynh. | AAB41504.1 | AtFLS |
| Fagopyrum tataricum (L.) Gaertn. | AEC33116.1 | FtFLS |
| Lilium regale E.H.Wilson | ASV46329.1 | LrFLS |
| Narcissus tazetta L. | AFS63899.1 | NtFLS |
| Ornithogalum longebracteatum Jacq. | QBQ58059.1 | OlFLS2 |
| Scutellaria baicalensis Georgi | AHA14501.1 | SbFLS |
Table 2 Gene information of system sequence alignment
| 拉丁名Latin name | 登录号Login ID | 基因名Gene name |
|---|---|---|
| Allium cepa L. | AAO63023.1 | AcFLS(H6) |
| A. cepa L. | AQR58516.1 | AcFLS (HRB) |
| Arabidopsis thaliana (L.) Heynh. | AAB41504.1 | AtFLS |
| Fagopyrum tataricum (L.) Gaertn. | AEC33116.1 | FtFLS |
| Lilium regale E.H.Wilson | ASV46329.1 | LrFLS |
| Narcissus tazetta L. | AFS63899.1 | NtFLS |
| Ornithogalum longebracteatum Jacq. | QBQ58059.1 | OlFLS2 |
| Scutellaria baicalensis Georgi | AHA14501.1 | SbFLS |
Table 3 Gene information of phylogenetic tree
| 拉丁名Latin name | 登录号Login ID | 基因名Gene name |
|---|---|---|
| Acacia confusa Merr. | AFA55179.1 | AcFLS |
| Allium cepa L. | AAO63023.1 | AcFLS (H6) |
| A. cepa L. | AQR58516.1 | AcFLS (HRB) |
| A. cepa L. | AAT68476.1 | AcFLS |
| Antirrhinum majus L. | ABB53382.1 | AmFLS |
| Arabidopsis thaliana (L.) Heynh. | AAB41504.1 | AtFLS |
| Camellia nitidissima | ADZ28516.1 | CnFLS |
| C. sinensis (L.) O. Ktze. | ABM88786.1 | CsFLS |
| Citrus unshiu Marcow. | BAA36554.1 | CuFLS |
| Cyclamen purpurascens | BBA27024.1 | CpurFLS2 |
| Eustoma exaltatum subsp. russellianum | AAF64168.1 | EeFLS |
| Fagopyrum tataricum (L.) Gaertn. | AEC33116.1 | FtFLS |
| F. tataricum (L.) Gaertn. | AGE13752.1 | FtFLS1 |
| Gentiana trifloral Pall. | BAK09226.1 | GtFLS |
| Ginkgo biloba L. | ACY00393.1 | GbFLS1 |
| Glycine max (Linn.) Merr. | BAF31231.1 | GmFLS1 |
| Lilium regale E.H.Wilson | ASV46329.1 | LrFLS |
| Malus×domestica Borkh. | AAX89401.1 | MdFLS(M9) |
| Narcissus tazetta | AFS63899.1 | NtFLS |
| Nicotiana tabacum L. | BAF96939.1 | NtFLS |
| Ornithogalum longebracteatum | QBQ58059.1 | OlFLS2 |
| Petroselinum crispum | AAP57395.1 | PcFLS |
| Petunia×hybrida E.Vilm | CAA80264.1 | PhFLS |
| Rosa rugosa Thunb. | AIS22436.1 | RrFLS |
| Scutellaria baicalensis Georgi | AHA14501.1 | SbFLS |
| Tricyrtis sp. Shinonome | BAU20368.1 | TrFLS |
| Tulipa fosteriana W.Irving | AHY20032.1 | TfFLS1 |
| Vaccinium corymbosum | AKJ87100.1 | VcFLS |
Table 3 Gene information of phylogenetic tree
| 拉丁名Latin name | 登录号Login ID | 基因名Gene name |
|---|---|---|
| Acacia confusa Merr. | AFA55179.1 | AcFLS |
| Allium cepa L. | AAO63023.1 | AcFLS (H6) |
| A. cepa L. | AQR58516.1 | AcFLS (HRB) |
| A. cepa L. | AAT68476.1 | AcFLS |
| Antirrhinum majus L. | ABB53382.1 | AmFLS |
| Arabidopsis thaliana (L.) Heynh. | AAB41504.1 | AtFLS |
| Camellia nitidissima | ADZ28516.1 | CnFLS |
| C. sinensis (L.) O. Ktze. | ABM88786.1 | CsFLS |
| Citrus unshiu Marcow. | BAA36554.1 | CuFLS |
| Cyclamen purpurascens | BBA27024.1 | CpurFLS2 |
| Eustoma exaltatum subsp. russellianum | AAF64168.1 | EeFLS |
| Fagopyrum tataricum (L.) Gaertn. | AEC33116.1 | FtFLS |
| F. tataricum (L.) Gaertn. | AGE13752.1 | FtFLS1 |
| Gentiana trifloral Pall. | BAK09226.1 | GtFLS |
| Ginkgo biloba L. | ACY00393.1 | GbFLS1 |
| Glycine max (Linn.) Merr. | BAF31231.1 | GmFLS1 |
| Lilium regale E.H.Wilson | ASV46329.1 | LrFLS |
| Malus×domestica Borkh. | AAX89401.1 | MdFLS(M9) |
| Narcissus tazetta | AFS63899.1 | NtFLS |
| Nicotiana tabacum L. | BAF96939.1 | NtFLS |
| Ornithogalum longebracteatum | QBQ58059.1 | OlFLS2 |
| Petroselinum crispum | AAP57395.1 | PcFLS |
| Petunia×hybrida E.Vilm | CAA80264.1 | PhFLS |
| Rosa rugosa Thunb. | AIS22436.1 | RrFLS |
| Scutellaria baicalensis Georgi | AHA14501.1 | SbFLS |
| Tricyrtis sp. Shinonome | BAU20368.1 | TrFLS |
| Tulipa fosteriana W.Irving | AHY20032.1 | TfFLS1 |
| Vaccinium corymbosum | AKJ87100.1 | VcFLS |
Fig.2 PCR products of the cDNA of LaFLS in L. auratum and LsFLS in L. speciosum var. gloriosoides M, DNA Marker; A1, A2, Product of the LaFLS amplification in cDNA; B1, B2, Product of the LsFLS amplification in cDNA.
Fig.2 PCR products of the cDNA of LaFLS in L. auratum and LsFLS in L. speciosum var. gloriosoides M, DNA Marker; A1, A2, Product of the LaFLS amplification in cDNA; B1, B2, Product of the LsFLS amplification in cDNA.
Fig.3 Nucleotide and amino acid sequences of LaFLS and LsFLS A, Nucleotide and amino acid sequences of LaFLS; B, Nucleotide and amino acid sequences of LsFLS. The frames show the differences between the nucleotide and amino acid sequences of LaFLS and LsFLS.
Fig.3 Nucleotide and amino acid sequences of LaFLS and LsFLS A, Nucleotide and amino acid sequences of LaFLS; B, Nucleotide and amino acid sequences of LsFLS. The frames show the differences between the nucleotide and amino acid sequences of LaFLS and LsFLS.
Fig.4 Amino acid multiple sequence alignment analysis of LaFLS and LsFLS The underlined position is the DIOX-N domain, and the red box represents the 2-ketoglutaric acid and iron (Ⅱ) dependent dioxygenase domain; DHQ substrate specific binding sites, 2-ketoglutaric acid and Fe2+ sites are indicated by black triangles, asterisks and arrows, respectively.
Fig.4 Amino acid multiple sequence alignment analysis of LaFLS and LsFLS The underlined position is the DIOX-N domain, and the red box represents the 2-ketoglutaric acid and iron (Ⅱ) dependent dioxygenase domain; DHQ substrate specific binding sites, 2-ketoglutaric acid and Fe2+ sites are indicated by black triangles, asterisks and arrows, respectively.
Fig.5 The phylogenetic tree analysis of FLS
Fig.5 The phylogenetic tree analysis of FLS
Fig.6 Analysis of FLS signal peptide
Fig.6 Analysis of FLS signal peptide
Fig.7 Trans-membrane domain analysis of FLS protein
Fig.7 Trans-membrane domain analysis of FLS protein
Fig.8 Hydrophilic-hydrophobic analysis of FLS protein
Fig.8 Hydrophilic-hydrophobic analysis of FLS protein
Fig.9 Subcellular localization analysis of LaFLS and LsFLS proteins
Fig.9 Subcellular localization analysis of LaFLS and LsFLS proteins
Fig.10 RT-PCR expression of FLS in L. auratum and L. speciosum var. gloriosoides different tissues 1, Upper leaf; 2, Lower leaf; 3, Style; 4, Stigma; 5, Ovary; 6, Perianth; 7, Anther; 8, Perianth; LaEF, Reference gene in L. auratum; LsTIP, Reference gene in L. speciosum var. gloriosoides.
Fig.10 RT-PCR expression of FLS in L. auratum and L. speciosum var. gloriosoides different tissues 1, Upper leaf; 2, Lower leaf; 3, Style; 4, Stigma; 5, Ovary; 6, Perianth; 7, Anther; 8, Perianth; LaEF, Reference gene in L. auratum; LsTIP, Reference gene in L. speciosum var. gloriosoides.
Fig.11 Expression of FLS in L. auratum (A) and L. speciosum var. gloriosoides (B) at different development stages of flower buds Different lowercase letters indicate significant differences (P<0.05).
Fig.11 Expression of FLS in L. auratum (A) and L. speciosum var. gloriosoides (B) at different development stages of flower buds Different lowercase letters indicate significant differences (P<0.05).
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